Cellulose and hemicelluloses are important industrial raw materials and a source of renewable energy. They can be degraded and used by numerous microorganisms, including bacteria, yeast and fungi, that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al, J. Biol. Chem., vol. 276, no. 26, pp. 24309-24314, Jun. 29, 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., Bioresource Tech. 77:193-196, 2001). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., Biotechnol. Gen. Engineer. Rev. vol. 14, pp. 365-414, 1997).
The physical structure and morphology of native celluloses are complex and the fine details of its structure have been difficult to determine experimentally. However, the chemical composition of cellulose is simple, consisting of D-glucose residues linked by beta-1,4-glycosidic bonds to form linear polymers with chains length of over 10.000 glycosidic residues.
In order to be efficient, the digestion of cellulose requires several types of enzymes acting cooperatively. For example, cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases ([β]-D-glucoside glucohydrolase; EC 3.2.1.21) (“BG”). (Knowles et al., TIBTECH 5, 255-261, 1987; Schulein, Methods Enzymol., 160, 25, pp. 234-243, 1988).
Endoglucanases act mainly on the amorphous parts of the cellulose fibre, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, the presence of a cellobiohydrolase in a cellulase system is required for efficient solubilization of crystalline cellulose (Suumakki, et al. Cellulose 7:189-209, 2000). Beta-glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides (Freer, J. Biol. Chem. vol. 268, no. 13, pp. 9337-9342, 1993).
As mentioned above, cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose, such that the cellulases are readily produced in large quantities via fermentation. Filamentous fungi play a special role since many yeasts, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose. See, e.g., Aro eta., 2001; Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, 1988; Wood et al., Methods in Enzymology, vol. 160, no. 9, pp. 87-116, 1988, and Coughlan, et al.
Exo-cellobiohydrolase II (Cellobiohydrolase II, or CBH 2) refer to the cellobiohydrolases which degrade cellulose by hydrolyzing the cellobiose from the reducing end of the cellulose polymer chains. The cellobiohydrolase II group belongs to the same EC group, that is EC 3.2. 1.91, as the cellobiohydrolase I group, the difference being that cellobiohydrolase I degrade cellulose by hydrolyzing the cellobiose from the non-reducing end of the cellulose polymer chains.
The efficient enzymatic degradation of biomass is a key factor for the development of an improved second generation for the production of biofuels and synthesis of platform chemicals or biopolymers from renewable sources. Currently the most efficient hydrolytic systems are originating from fungi like Trichoderma reesei. They consist out of a mixture of enzymes (Minimal Enzyme Complex; MEC) which act complementary to depolymerize cellulose or hemicellulose to sugar monomers.
It is an object of the present invention to provide improved polypeptides having cellobiohydrolase II activity and polynucleotides encoding these polypeptides. The improved polypeptides may have improved thermostability and/or improved stability, but in particular improved specific activity.